The new title of this revised application: "beta- synuclein as a treatment for Lewy body disease" reflects the modified and sharper focus on our scientific approach in response to the reviewers' major concern that synuclein might be more relevant for understanding Lewy body disease (LBD). We now propose a new concept where beta-synuclein, a naturally occurring anti-aggregation molecule and non-amyloidogenic homologue of alpha-synuclein, might prevent the neurotoxic effects of alpha- synuclein and could be a suitable target for the development of an alternative treatment for LBD. In this context, we propose the following Specific Aims: 1) To characterize the mechanisms by which beta-synuclein blocks alpha-synuclein aggregation. We hypothesize that beta-synuclein may interact with alpha-synuclein through specific beta-synuclein domains, leading to inhibition of alpha-synuclein aggregation. To test this hypothesis, we will study the effects of mutant recombinant beta-synucleins and synthetic beta-synuclein derived peptides on alpha-synuclein aggregation, using immunoblotting analysis, Congo red/Thioflavine-S staining, and electron microscopy. Additional studies of synuclein binding will be performed by immunoblotting with His-tagged alpha- and beta-synuclein 2) To determine if the anti-aggregation effect of beta-synuclein is protective in neuronal cell lines expressing alpha-synuclein. We hypothesize that beta-synuclein may be neuroprotective by blocking alpha- synuclein aggregation. To test this hypothesis, alpha-synuclein- overexpressing GT1-7 and B103 neuronal cells will be co- transfected with beta-synuclein GT1-7 cells will be evaluated for cell viability, mitochondrial function, oxidative stress conditions, GnRH secretion, altered mitochondria morphology and inclusion body formation. B103 cells will be evaluated by analysis of neurite formation and cell adhesion. Immunoblotting experiments will be performed with cells co-transfected with c- myc-tagged beta-synuclein to assess binding to alpha-synuclein. To determine potential novel treatments, alpha-synuclein- overexpressing GT1-7 and B103 cells will be treated with beta- synuclein-expressing recombinant adeno-associated viral vector (rAAV). 3) To determine if beta-synuclein blocks alpha-synuclein aggregation and neurodegeneration in in vivo model systems of LBD. We hypothesize that beta-synuclein may inhibit alpha- synuclein aggregation in vivo. To test this hypothesis, we will cross alpha-synuclein tg mice with either beta- synuclein tg or knockout mice where murine alpha- or beta-synuclein gene is deleted. Mice will undergo detailed behavioral, neurochemical and neuropathological examination to determine if beta-synuclein expression affects the functional and structural alterations promoted by alpha-synuclein and might act as a basis for treatment of LBD. To assess the potential for treatment development, alpha-synuclein tg mice will be treated with a beta- synuclein-expressing rAAV. In summary, we will utilize a multi-system approach (a cell-free, cell culture and tg mouse systems) to ascertain the anti- aggregation potential of beta-synuclein as a therapeutic target for development of novel treatments for LBD.